Stabilized α-galactosidase and uses thereof

Abstract

Multimeric protein structures comprising at least two alpha-galactosidase monomers being covalently linked to one another via a linking moiety are disclosed herein, as well a process for preparing same, and methods of treating Fabry disease via administration of a multimeric protein structure. The disclosed multimeric protein structures exhibit an improved performance, in terms of enhanced activity and/or a longer lasting activity under both lysosomal conditions and in a serum environment.

What is claimed is: 
     
       1. A multimeric protein structure comprising at least two α-galactosidase A monomers being covalently linked to one another via a linking moiety, the multimeric protein structure featuring a characteristic selected from the group consisting of:
 (a) an α-galactosidase activity upon subjecting the multimeric protein structure to human plasma conditions for one hour, which is at least 10% higher than an activity of native α-galactosidase A upon subjecting said native α-galactosidase A to said human plasma conditions for one hour; 
 (b) an α-galactosidase activity which decreases upon subjecting the multimeric protein structure to human plasma conditions for one hour by a percentage which is at least 10% less than the percentage by which an activity of said native α-galactosidase A decreases upon subjecting said native α-galactosidase A to said human plasma conditions for one hour; 
 (c) an α-galactosidase activity which remains substantially unchanged upon subjecting the multimeric protein structure to human plasma conditions for one hour; 
 (d) an α-galactosidase activity, upon subjecting the multimeric protein structure to lysosomal conditions for one week, which is at least 10% higher than an activity of native α-galactosidase A upon subjecting said native α-galactosidase A to said lysosomal conditions for one week; 
 (e) an α-galactosidase activity which decreases upon subjecting the multimeric protein structure to lysosomal conditions for one day by a percentage which is at least 10% less than the percentage by which an activity of said native α-galactosidase A decreases upon subjecting said native α-galactosidase A to said lysosomal conditions for one day; 
 (f) an α-galactosidase activity which remains substantially unchanged upon subjecting the multimeric protein structure to lysosomal conditions for one day; 
 (g) an α-galactosidase activity, immediately upon subjecting the multimeric protein structure to lysosomal conditions, which is at least 10% higher than an activity of native α-galactosidase A immediately upon subjecting said native form of said protein to said lysosomal conditions; 
 (h) an α-galactosidase activity, immediately upon subjecting the multimeric protein structure to an aqueous solution having a pH of 7 and a temperature of 37° C., which is at least 10% higher than an activity of native α-galactosidase A immediately upon subjecting said native α-galactosidase A to said aqueous solution having a pH of 7 and a temperature of 37° C.; and 
 (i) a circulating half-life in a physiological system which is higher by at least 20% than said circulating half-life of said native α-galactosidase A. 
 
     
     
       2. A process of preparing the multimeric protein structure of  claim 1 , the process comprising reacting α-galactosidase A with a cross-linking agent which comprises said linking moiety and at least two reactive groups. 
     
     
       3. A multimeric protein structure comprising at least two α-galactosidase A monomers being covalently linked to one another via a linking moiety, wherein said linking moiety is not present in native α-galactosidase A. 
     
     
       4. A process of preparing the multimeric protein structure of  claim 3 , the process comprising reacting α-galactosidase A with a cross-linking agent which comprises said linking moiety and at least two reactive groups. 
     
     
       5. A multimeric protein structure comprising two α-galactosidase monomers, said at least two α-galactosidase monomers being α-galactosidase A monomers, the protein structure being a dimeric protein structure, said two α-galactosidase monomers being covalently linked to one another via a linking moiety comprising a poly(alkylene glycol). 
     
     
       6. The multimeric protein structure of  claim 5 , wherein said poly(alkylene glycol) comprises at least two functional groups, each functional group forming a covalent bond with one of the α-galactosidase monomers. 
     
     
       7. The multimeric protein structure of  claim 6 , wherein said at least two functional groups are terminal groups of said poly(alkylene glycol). 
     
     
       8. The multimeric protein structure of  claim 6 , wherein at least one of said functional groups forms an amide bond with an α-galactosidase monomer. 
     
     
       9. The multimeric protein structure of  claim 5 , wherein said poly(alkylene glycol) comprises at least 5 alkylene glycol units. 
     
     
       10. The multimeric protein structure of  claim 5 , wherein said poly(alkylene glycol) is poly(ethylene glycol). 
     
     
       11. The multimeric protein structure of  claim 5 , featuring a characteristic selected from the group consisting of:
 (a) an α-galactosidase activity, upon subjecting the multimeric protein structure to human plasma conditions for one hour, which is at least 10% higher than an activity of native α-galactosidase A upon subjecting said native α-galactosidase A to said human plasma conditions for one hour; 
 (b) an α-galactosidase activity which decreases upon subjecting the multimeric protein structure to human plasma conditions for one hour by a percentage which is at least 10% less than the percentage by which an activity of said native α-galactosidase A decreases upon subjecting said native α-galactosidase A to said human plasma conditions for one hour; 
 (c) an α-galactosidase activity which remains in a range of 50% to 150% of the initial activity upon subjecting the multimeric protein structure to human plasma conditions for one hour; 
 (d) an α-galactosidase activity, upon subjecting the multimeric protein structure to lysosomal conditions for one week, which is at least 10% higher than an activity of native α-galactosidase A upon subjecting said native α-galactosidase A to said lysosomal conditions for one week; 
 (e) an α-galactosidase activity which decreases upon subjecting the multimeric protein structure to lysosomal conditions for one day by a percentage which is at least 10% less than the percentage by which an activity of said native α-galactosidase A decreases upon subjecting said native α-galactosidase A to said lysosomal conditions for one day; 
 (f) an α-galactosidase activity which remains in a range of 50% to 150% of the initial activity upon subjecting the multimeric protein structure to lysosomal conditions for one day; 
 (g) an α-galactosidase activity, immediately upon subjecting the multimeric protein structure to lysosomal conditions, which is at least 10% higher than an activity of native α-galactosidase A immediately upon subjecting said native α-galactosidase A to said lysosomal conditions; 
 (h) an α-galactosidase activity, immediately upon subjecting the multimeric protein structure to an aqueous solution having a pH of 7 and a temperature of 37° C., which is at least 10% higher than an activity of native α-galactosidase A immediately upon subjecting said native α-galactosidase A to said aqueous solution having a pH of 7 and a temperature of 37° C.; and 
 (i) a circulating half-life in human plasma which is higher than a circulating half-life of said native α-galactosidase A. 
 
     
     
       12. The multimeric protein structure of  claim 5 , wherein said α-galactosidase is a human α-galactosidase selected from the group consisting of agalsidase alpha and agalsidase beta. 
     
     
       13. The multimeric protein structure of  claim 5 , wherein said α-galactosidase is a plant recombinant α-galactosidase. 
     
     
       14. The multimeric protein structure of  claim 5 , wherein said α-galactosidase comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 15. 
     
     
       15. The multimeric protein structure of  claim 5 , wherein said α-galactosidase has an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO: 15. 
     
     
       16. A pharmaceutical composition comprising the multimeric protein structure of  claim 5  and a pharmaceutically acceptable carrier. 
     
     
       17. A method of treating Fabry disease, the method comprising administering to a subject in need thereof a therapeutically effective amount of the multimeric protein structure of  claim 5 , thereby treating the Fabry disease. 
     
     
       18. The method of  claim 17 , wherein said administering is effected by intravenous infusion. 
     
     
       19. A process of preparing the multimeric protein structure of  claim 5 , the process comprising reacting α-galactosidase A with a cross-linking agent which comprises said linking moiety and at least two reactive groups. 
     
     
       20. The process of  claim 19 , wherein said reactive groups comprise a leaving group.